Microbiology Lab Study Guide: Physiological Tests, Staphylococcus & Streptococcus, White Blood Cells, and Epidemiology

Physiological Characteristics of Microorganisms

Overview of Biochemical Tests

Microbiologists use a variety of biochemical tests to identify and characterize bacteria based on their metabolic activities. These tests often detect the presence of specific enzymes, metabolic products, or the ability to utilize certain substrates. Understanding these tests is essential for interpreting laboratory results and distinguishing between bacterial species.

• Sugar Fermentation: Tests the ability of bacteria to ferment sugars, producing acid and sometimes gas. - Media: Phenol red sugar broth - Enzyme: Varies by sugar - Control Microorganism: Escherichia coli (glucose) - Positive Result: Acid (yellow color); gas (bubble in Durham tube) - Notes: Gas may not always be present.

• Mixed Acid Fermentation (Methyl Red Test): Detects production of stable acids from glucose fermentation. - Media: MR-VP broth - Enzyme: Formic hydrogenylase - Control: Escherichia coli - Reagent: Methyl red - Positive Result: Red color after reagent addition

• Butanediol Fermentation (Voges-Proskauer Test): Detects neutral end products (acetoin) from glucose fermentation. - Media: MR-VP broth - Control Microorganism: Enterobacter aerogenes - Reagents: Barritt's A & B - Positive Result: Red color with aeration (takes ~10 min)

• Nitrate Reduction: Tests for reduction of nitrate to nitrite or nitrogen gas. - Media: Nitrate broth - Enzyme: Nitratase (nitrate reductase) - Control: Escherichia coli - Reagents: Nitrite test reagents (A & B) - Positive Result: Red color (may fade)

• Starch Hydrolysis: Detects breakdown of starch by amylase. - Media: Starch agar - Enzyme: Amylase - Control: Bacillus subtilis - Reagent: Gram's iodine - Positive Result: Clearing around growth

• Casein Hydrolysis: Tests for breakdown of casein protein by caseinase. - Media: Skim milk agar - Enzyme: Caseinase - Control: Bacillus subtilis - Positive Result: Clearing around growth

• Fat (Lipid) Hydrolysis: Detects breakdown of lipids by lipase. - Media: Spirit blue agar - Enzyme: Lipase - Control: Staphylococcus aureus - Positive Result: Clearing and blue precipitate

• Tryptophan Hydrolysis (Indole Test): Tests for breakdown of tryptophan to indole by tryptophanase. - Media: Tryptose broth or SIM medium - Enzyme: Tryptophanase - Control: Escherichia coli - Reagent: Kovac's - Positive Result: Red ring/layer on top

• Urea Hydrolysis: Detects breakdown of urea by urease. - Media: Urea broth - Enzyme: Urease - Control: Proteus vulgaris - Positive Result: Hot pink (fuchsia) color

• Phenylalanine Deamination: Tests for removal of amino group from phenylalanine by phenylalanase. - Media: Phenylalanine agar slant - Enzyme: Phenylalanase - Control: Proteus vulgaris - Reagent: Ferric chloride - Positive Result: Green slant

• Hydrogen Sulfide (H2S) Production: Detects production of H2S from cysteine by cysteine desulfurase. - Media: SIM medium - Enzyme: Cysteine desulfurase - Control: Proteus vulgaris - Positive Result: Black precipitate

• Citrate Utilization: Tests for use of citrate as sole carbon source by citratase. - Media: Simmon's citrate agar slant - Enzyme: Citratase - Control: Enterobacter aerogenes - Positive Result: Growth and blue color

• Catalase Production: Detects catalase enzyme that breaks down hydrogen peroxide. - Media: Any solid medium - Enzyme: Catalase - Control: Staphylococcus aureus - Reagent: Hydrogen peroxide - Positive Result: Bubbles

• Oxidase Production: Tests for cytochrome oxidase enzyme. - Media: Any solid medium (e.g., TSA) - Enzyme: Oxidase - Control: Pseudomonas aeruginosa - Reagent: Oxidase test reagent - Positive Result: Purple color on cotton swab within 2 min

Summary Table: Biochemical Tests

Test Name	AKA	Medium	Enzyme	Control M/O	Positive Result	Reagent
Sugar Fermentation	-	Phenol red sugar broth	Varies	Escherichia coli	Yellow (acid), gas (bubble)	-
Mixed Acid Fermentation	Methyl Red	MR-VP	Formic hydrogenylase	Escherichia coli	Red	Methyl red
Butanediol Fermentation	Voges-Proskauer	MR-VP	-	Enterobacter aerogenes	Red (with aeration)	Barritt's A & B
Nitrate Reduction	-	Nitrate broth	Nitratase	Escherichia coli	Red	Nitrite reagents
Starch Hydrolysis	-	Starch agar	Amylase	Bacillus subtilis	Clearing	Gram's iodine
Casein Hydrolysis	-	Skim milk agar	Caseinase	Bacillus subtilis	Clearing	-
Fat Hydrolysis	-	Spirit blue agar	Lipase	Staphylococcus aureus	Clearing, blue ppt.	-
Tryptophan Hydrolysis	Indole test	Tryptose broth/SIM	Tryptophanase	Escherichia coli	Red ring	Kovac's
Urea Hydrolysis	-	Urea broth	Urease	Proteus vulgaris	Hot pink	-
Phenylalanine Deamination	-	Phenylalanine agar slant	Phenylalanase	Proteus vulgaris	Green slant	Ferric chloride
H2S Production	-	SIM medium	Cysteine desulfurase	Proteus vulgaris	Black ppt.	-
Citrate Utilization	-	Simmon's citrate agar	Citratase	Enterobacter aerogenes	Blue	-
Catalase Production	-	Any solid medium	Catalase	Staphylococcus aureus	Bubbles	H2O2
Oxidase Production	-	Any solid medium	Oxidase	Pseudomonas aeruginosa	Purple (cotton swab)	Oxidase reagent

Staphylococcus: Characteristics and Identification

Overview of the Genus Staphylococcus

Staphylococcus is a genus of Gram-positive cocci found in clusters. Members of this genus are important in clinical microbiology due to their role in human disease, especially nosocomial (hospital-acquired) infections.

• Family: Staphylococcaceae

• Pyogenic: Pus-forming

• Diseases: Abscesses, boils, wound infections, toxic shock syndrome

• Habitat: Nasal membranes, hair follicles, skin

• Catalase Positive: Distinguishes from Streptococcus (catalase negative)

• MRSA: Methicillin-resistant Staphylococcus aureus (major nosocomial pathogen)

• Medically Significant Species: S. aureus (most pathogenic), S. epidermidis, S. saprophyticus

Identification Methods

• Mannitol Salt Agar (MSA): - Selective: High NaCl inhibits Gram negatives and non-halophiles - Differential: Contains mannitol and phenol red; fermentation turns plate yellow - Results: S. aureus ferments mannitol (yellow), S. epidermidis does not (no color change)

• Hemolysis on Blood Agar: - Beta: Complete destruction of RBCs (clear zone) - Alpha: Partial destruction (greenish zone) - Gamma: No hemolysis - S. aureus is beta hemolytic

• Coagulase Test: - S. aureus produces coagulase, which clots plasma - Procedure: Inoculate plasma, incubate 24 hr; positive = clotted plasma

Streptococcus: Characteristics and Identification

Overview of the Genus Streptococcus

Streptococcus is a genus of Gram-positive cocci found in chains. These bacteria are significant pathogens, causing a variety of diseases in humans.

• Family: Streptococcaceae

• Can be Pyogenic: Pus-forming

• Diseases: Pneumonia, meningitis, pharyngitis, endocarditis, glomerulonephritis

• Habitat: Pharyngeal cavity, teeth, saliva, colon

• Catalase Negative: Distinguishes from Staphylococcus

• Medically Significant Species: S. pyogenes, S. agalactiae, S. pneumoniae, Enterococcus

Lancefield Groupings

Rebecca Lancefield classified Streptococcus species based on their cell wall antigens, assigning them to groups labeled with letters (A, B, C, D, etc.).

Identification Methods

• Hemolysis: - Beta: Complete - Alpha: Partial - Gamma: None

• Bacitracin Susceptibility: - Beta hemolytic strep: Any zone = positive for S. pyogenes (Group A)

• SXT Susceptibility: - Beta hemolytic strep: Any zone = positive for Group C

• CAMP Test: - Beta hemolytic strep: Arrowhead-shaped zone of hemolysis where Group B intersects with S. aureus - S. agalactiae is positive

• Bile Esculin Hydrolysis: - Alpha hemolytic strep: Blackening of slant = Group D

• Salt Tolerance: - Alpha hemolytic strep: Turbidity within 72 hr = Group D enterococci

• Optochin Susceptibility: - Alpha hemolytic strep: Measured zone; S. pneumoniae is positive

Comparison Table: Staphylococcus vs. Streptococcus

Feature	Staphylococcus	Streptococcus
Gram Reaction	Positive	Positive
Shape	Cocci in clusters	Cocci in chains
Catalase	Positive	Negative
Hemolysis	Beta (S. aureus)	Alpha, Beta, Gamma
Diseases	Abscesses, boils, TSS	Pneumonia, pharyngitis

White Blood Cell Study

Types and Identification of White Blood Cells (WBCs)

White blood cells are crucial components of the immune system. They are classified as granulocytes or agranulocytes based on the presence of granules in their cytoplasm. Normal percentages and identification features are important for clinical diagnosis.

• Granulocytes:

  • Neutrophils: 50-70% (multi-lobed nucleus, pale granules)

  • Eosinophils: 1-5% (bi-lobed nucleus, red-orange granules)

  • Basophils: <1% (bi-lobed nucleus, dark blue granules)

• Agranulocytes:

  • Lymphocytes: 20-30% (large, round nucleus, scant cytoplasm)

  • Monocytes: 2-6% (kidney-shaped nucleus, abundant cytoplasm)

• Identification: Size compared to RBCs, nucleus size/shape, granulation/color

Normal WBC Percentages Table

WBC Type	Normal %
Neutrophils	50-70%
Eosinophils	1-5%
Basophils	<1%
Lymphocytes	20-30%
Monocytes	2-6%

Synthetic Epidemic and Epidemiology

Key Concepts in Epidemiology

Epidemiology is the study of disease distribution and spread in populations. Understanding the terminology and historical cases is essential for recognizing patterns and controlling outbreaks.

• Infectious Disease: Caused by microorganisms that multiply in host tissues; communicable

• Epidemiology: Study of disease spread/distribution

• Epidemic: Excessive new cases in a specific area/time

• Endemic: Steady frequency in a region

• Pandemic: Disease spreads across continents

• Types of Epidemics: Common source (single origin), Propagated (host-to-host)

Case Study: "Typhoid Mary" (Mary Mallon)

• Who: Head cook in New York City

• What: Spread Salmonella typhi (Typhoid Fever)

• When: Late 1800s, early 1900s

• How: Asymptomatic carrier; shed bacteria from gall bladder via fecal material; poor hygiene led to transmission

Additional info:

• Abnormal WBC counts can indicate infection, allergy, or hematologic disorders.

• IMViC reactions (Indole, Methyl Red, Voges-Proskauer, Citrate) are a set of tests used to differentiate Enterobacteriaceae.